It is already known that many kinds of microorganisms accumulate polyesters as the energy-storing substance intracellularly. A representative example is a homopolymer of 3-hydroxybutyric acid (hereinafter referred to briefly as 3HB), namely poly-3-hydroxybutyric acid (hereinafter referred to briefly as P(3HB)), which was first discovered in Bacillus megaterium in 1925. Since P(3HB) is a thermoplastic polymer biodegradable in the natural environment, it has attracted attention as an eco-friendly plastic. However, because of its high crystallinity, P(3HB) is so hard and brittle that it has found application so far only in a limited assortment of practical uses and much research work has been undertaken to correct for the drawback.
As results of such researches, Japanese Kokai Publication Sho-57-150393 and Japanese Kokai Publication Sho-59-220192 disclose the production technology for a copolymer of 3-hydroxybutyric acid (3HB) and 3-hydroxyvaleric acid (3HV) (hereinafter referred to briefly as P(3HB-co-3HV)). Compared with P(3HB), this P (3HB-co-3HV) is so flexible that it was initially expected to find application in a broader range of end uses. Actually, however, P(3HB-co-3HV) responds poorly to a gain in the molar fraction of 3HV so that particularly the flexibility required of film and the like cannot be improved, thus limiting its scope of use to rigid moldings such as shampoo bottles and throw-away razor handles.
Recently, studies have been undertaken on the binary copolyester of 3HB and 3-hydroxyhexanoic acid (hereinafter referred to briefly as 3HH) (the copolyester will hereinafter be referred to briefly as P(3HB-co-3HH)) and the technology of producing it. For example, such studies have been described in Japanese Kokai Publication Hei-5-93049 and Japanese Kokai Publication Hei-7-265065. The technology described in the above patent literature for the production of P(3HB-co-3HH) comprises fermentative production from a fatty acid, e.g. oleic acid, or an oil or fat, e.g. olive oil, with the aid of a soil-isolated strain of Aeromonas caviae. Properties of P(3HB-co-3HH) have also been studied [Y. Doi, S. Kitamura, H. Abe: Macromolecules 28, 4822–4823 (1995)]. In this report referred to above, Aeromonas caviae is cultured using a fatty acid of 12 or more carbon atoms as the sole carbon source to fermentatively produce P(3HB-co-3HH) with a 3HH fraction of 11 to 19 mol %. This P(3HB-co-3HH) undergoes a gradual transition from a hard, brittle one to a flexible one with an increasing molar fraction of 3HH and has been found to show flexibility surpassing that of P(3HB-co-3HV). However, this production method is poor in productivity with a cell output of 4 g/L and a polymer content of 30% and, therefore, explorations were made for methods of higher productivity for commercial exploitation.
A PHA (polyhydroxyalkanoic acid)-synthase gene was cloned from a P(3HB-co-3HH)-producible strain of Aeromonas caviae [T. Fukui, Y. Doi: J. Bacteriol, Vol. 179, No. 15, 4821–4830 (1997), Japanese Kokai Publication Hei-10-108682]. When this gene was introduced into Ralstonia eutropha (formerly, Alcaligenes eutrophus) and the production of P(3HB-co-3HH) was carried out using the resulting transformant, the cell output was 4 g/L and the polymer content was 30%. Further, by growing this transformant on vegetable oil as the carbon source, a cell content of 4 g/L with a polymer content of 80% could be accomplished [T. Fukui et al.: Appl. Microbiol. Biotechnol. 49, 333 (1998)]. A process for producing P(3HB-co-3HH) using bacteria, e.g. Escherichia coli, or plants as hosts has also been described (WO 00/43525). However, there is no disclosure of the productivity achieved by this production technology.
Since this polymer P(3HB-co-3HH) may have a broadly variable characteristic ranging from a rigid polymer to a flexible polymer depending on the 3HH molar fraction, it can be expected to find application in a broad spectrum of uses from television housings and the like, which require rigidity, to yarn, film and the like which require flexibility. However, the productivity of said polymer is still invariably low in these production methods and none are considered fully satisfactory for practical production methods of this polymer.
Recently, Leaf et al. have conducted studies on the production of biodegradable polyesters using a yeast, which is considered to elaborate acetyl CoA, the precursor of 3HB, with good efficiency as a producer organism (Microbiology, Vol. 142, pp 1169–1180 (1996)). They introduced the Ralstonia eutropha polyester synthase gene into Saccharomyces cerevisiae, a kind of yeast, to construct a transformant and cultured it using glucose as the carbon source, to thereby confirm the accumulation of P(3HB) (polymer content 0.5%). However, the polymer produced in this study was P (3HB), which is hard and brittle.
It is known that yeasts are fast-growing, with high cell productivity. The yeast cell attracted attention as the single cell protein in the past and studies on the production of yeast cells for use as a feedstuff using n-paraffin as the carbon source, while their component nucleic acids have been utilized as seasonings. Furthermore, since yeasts are considered to produce acetyl-CoA, which is a precursor of the polymer, with high efficiency, a high polymer productivity is expected. Moreover, since the separation of cells from the culture broth is easy as compared with bacteria, it is possible to simplify the polymer extraction and purification process. Therefore, a demand has existed for a process for producing P(3HB-co-3HH) having beneficial physical properties with the aid of yeasts.